The aim of this proposal is to study the regulatory mechanisms of liver cell proliferation using primary cultures of adult rat liver parenchymal cells. Our previous studies defined hormonal conditions for the stimulation of DNA replication and cell division in cells isolated from partially hepatectomized livers. Combinations of insulin, epidermal growth factor (EGF) and glucagon, at physiological concentrations, acted synergistically to stimulate incorporation of labeled thymidine into morphologically distinct hepatocyte nuclei. We have now extended these studies to cells from intact rat livers. We have optimized conditions for culturing these cells and find similar hormonal requirements as with the cells from partially hepatectomized livers. In the presence of the 3 hormones the cultured cells enter DNA synthesis on the third day and peak on the fourth day. The glucagon requirement can be replaced by cAMP or catacholamines. All three hormones are required on the first day of culture for optimal synthesis; EGF and insulin are also required after the first day. Further studies will be carried out into the early event in the hormonal stimulation of cell growth. Insulin, EGF and glucagon will be further characterized to determine if they act to initiate the growth process or to promote progression towards DNA synthesis. We will also determine the effects of hormone mixtures and of the individual hormones on RNA synthesis, and on the ability to regulate nuclear protein phosphorylation.